1. Field of the Invention
The present invention relates to a method for producing L-cysteine, more precisely a novel coryneform bacterium suitable for the production of L-cysteine and a method for producing L-cysteine utilizing the bacterium. L-Cysteine and L-cysteine derivatives are used in the fields of drugs, cosmetics and foods.
2. Description of the Related Art
L-Cysteine is conventionally obtained by extracting it from keratin-containing materials such as hairs, horns and feathers or microbial enzymatic conversion utilizing DL-2-aminothiazoline-4-carboxylic acid as a precursor. Furthermore, it is also attempted to produce L-cysteine by fermentation utilizing a microorganism.
The biosynthesis of L-cysteine have been studied in detail for bacteria such as Escherichia coli (Kredich, N. M. et al., J. Biol. Chem., 241, 4955–4965 (1966); Kredich, N. M. et al., 1987, Biosynthesis of Cysteine, In: Neidhardt, F. C., et al. (eds), Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, Vol.1, American Society for Microbiology, Washington D.C., 419–428), and it has been elucidated that L-cysteine is produced from L-serine through reactions of two steps. In Escherichia coli, the first reaction is activation of L-serine by acetyl-CoA, and it is catalyzed by serine acetyltransferase (EC 2.3.1.30, also abbreviated as “SAT” hereinafter). The second reaction is a reaction by which L-cysteine is produced from O-acetylserine that is produced by the above reaction, and it is catalyzed by O-acetylserine (thiol) lyase.
Further, it is also known that, in Escherichia coli, decomposition of L-cysteine is suppressed by reduction of cysteine desulfhydrase (also abbreviated as “CD” hereinafter) activity (Japanese Patent Laid-open Publication (Kokai) No. 11-155571).
In Escherichia coli, genes coding for SAT (cysE) have been cloned from a wild strain and an L-cysteine secreting mutant strain (Denk, D. and Boeck, A., J. General Microbiol., 133, 515–525 (1987)). Further, the nucleotide sequences of these cysE have been determined, and it has been reported that the methionine residue at a position of 256 is replaced with an isoleucine residue in SAT of which feedback inhibition by L-cysteine is decreased. Furthermore, there has also been disclosed a method of producing L-cysteine or the like by using a DNA coding for SAT of which feedback inhibition by L-cysteine is reduced by a mutation different from the aforementioned mutation (International Patent Publication WO97/15673). This SAT has a mutation in the region from the 97th amino acid residue to the 273rd amino acid residue, or a deletion of a C-terminus region from the 227th amino acid residue.
While there were known techniques for producing L-cysteine by utilizing a gene coding for SAT of which feedback inhibition by L-cysteine was reduced as described above, the inventors of the present invention found that productivity of L-cysteine of a bacterium belonging to the genus Escherichia containing SAT of which feedback inhibition by L-cysteine was reduced was unstable. And they also found that this instability could be eliminated, i.e., the productivity could be stabilized, by reducing the intracellular CD activity, and they successfully created an Escherichia coli strain that stably produced L-cysteine (Japanese Patent Laid-open Publication No. 11-155571).
Further, there has also been disclosed a method for producing L-cysteine and so forth by using a microorganism, specifically Escherichia coli, that excessively expresses a gene coding for a protein suitable for discharging antibiotics or other toxic substances from a cell (gene for excretion) (Japanese Patent No. 29920110).
On the other hand, as for Corynebacterium glutamicum, the aecD gene has been identified as a gene that imparts S-(β-aminoethyl)-cysteine (AEC) resistance (Rossol, I. et al., J. Bacteriol., 174 (9), 2968–2977 (1992)). It has been demonstrated that the aecD gene is not indispensable for growth, it is involved in the AEC resistance only when it is amplified, and a protein encoded by this gene has C-S lyase activity of which substrate is AEC, cysteine or the like. Rossol et al. paid attention to the fact that lyase could catalyze elimination reaction and reverse synthetic reaction like synthetase, and suggested novel enzymatic synthesis of sulfur-containing amino acids. However, since any example of successful production of sulfur-containing amino acids by fermentation using a microorganism had scarcely been known, they mentioned only an enzymatic synthesis method using the reverse reaction as for possible use of the C-S lyase.
As described above, there are several reports about the breeding of L-cysteine producing bacteria belonging to the genus Escherichia, and enzymes involved in the L-cysteine decomposition of Corynebacterium glutamicum have also been reported to a certain extent. However, any example of L-cysteine production by fermentation to such an extent that L-cysteine can be collected from a medium has not been known for coryneform bacteria.